Our research is focused on elucidating the basic mechanisms by which selenoproteins are synthesized in vivo and to investigate the structure and function of selenoenzymes. Currently, we are investigating the following projects:[unreadable] [unreadable] (a) Selenophosphate is the selenium donor for the biosynthesis of selenocysteine-containing proteins and seleno-tRNA. Selenophosphate synthetase (SPS) catalyzes the formation of selenophosphate from ATP and selenide. The structural study of SPS has been hampered by the difficulty in obtaining suitable crystals for x-ray crystallographic analysis. We have now successfully crystallized a SPS (C17S) mutant in which selenomethionine is substituted for methionine. The structural analysis revealed that the SPS mutant exists a homodimer with a flexible N-terminal region. A wild-type SPS crystal is also being prepared for structural studies. Furthermore, in vitro kinetic studies revealed a selenide Km of 7.3 microM for SPS, which is significantly higher than the toxic level for mammals. Therefore, it is reasonable to assume the presence of a component capable of lowering its Km value or the existence of a selenium delivery protein, such that the SPS can function with a non-toxic level of selenide. To this end, we have investigated various potential candidates, including glyceraldehye-3-phosphate dehydrogenase, a well-known glycolytic enzyme that has been shown to bind selenium, and a novel selenium-binding protein (SeBP) isolated from Methanococcus vannielii. Both proteins contain a low pKa cysteine as the selenium binding site. In both cases, the selenium donor is selenodiglutathione, GSSeSG, the intermediate in the reduction of selenite by GSH. To better understand how SeBP works as a selenium-binding protein, the solution structure of SeBP was determined by Nuclear Magnetic Resonnance (NMR) methods. The monomer of SeBP is composed of an alpha-helix on top of a twisted beta-sheet formed by four beta-strands. However, in solution, SeBP forms a stable pentamer.[unreadable] [unreadable] (b) Initial studies on selenium metabolism in a single-celled eukaryotic organism, the amoeba form of Dictyostelium, revealed the presence of about three to four selenium-containing proteins based on the presence of 75-Se-labeled protein bands. To identify these proteins, a large-scale preparation is being carried out to provide larger protein samples for 2D gel separation and proteomic analysis.[unreadable] [unreadable] (c) Overexpression of selenium-containing proteins is hampered by the requirement of a complex co-translational selenocysteine incorporation mechanism. To bypass this problem, we collaborated with Peter Schultz to use his methodology to incorporate unnatural amino acid into a specific site of a given protein in both prokaryotic and eukaryotic organisms. This method relies on a unique codon-tRNA pair and corresponding aminoacyl tRNA synthetase for the unnatural amino acid that do not cross-react with any of the endogenous tRNAs, aminoacyl tRNA synthetases, amino acids, or codons in the host organism. Initially, we chose three selenoproteins--methionine sulfoxide reductase B1, SelW protein, and thioredoxin reductase 1 since we can verify the integrity of the synthesized product by monitoring the specific activity of both selenoenzymes and SelW in the smallest selenoprotein. We are currently working with the E. coli expression system.